• Gaussian blur sigma=2, convert to frequencies before pixel FlowSOM
  • Pixel hClusters found using z-score cap=3, k=20, and iterative thresh=2 (1 iteration)
  • Cell-based clustering using frequency of 100 pixel FlowSOM clusters, 23 hCluster (iter), normalize by size, k=20

1 Histogram of counts in pixel clusters

2 Heatmaps of pixel clusters x markers

3 Compare pixel clusering with cell clustering

4 Cell-based FlowSOM clustering using pixel clusters

4.1 Cell cluster x frequency of pixel clusters

4.2 Cell cluster x marker (not weighted, segmentation)

4.3 Cell cluster x marker (weighted by pixel cluster frequency)

5 Cell-based FlowSOM clustering using hClusters (cap, k=20)

5.1 Cell cluster x frequency of pixel clusters

5.2 Cell cluster x marker (not weighted, segmentation)

5.3 Cell cluster x marker (weighted by pixel cluster frequency)

6 Cell-based FlowSOM clustering using hClusters (iter, k=23)

6.1 Cell cluster x frequency of pixel clusters

6.2 Cell cluster x marker (not weighted, segmentation)

6.3 Cell cluster x marker (weighted by pixel cluster frequency)

7 Heatmaps from normal segmentation